Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 angstrom by a tyrosyl radical (Y-122, Escherichia coli) in subunit beta of a cysteine (C-439) in the active site of subunit a. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y-122 reversible arrow Y-356 in beta to Y-731 reversible arrow Y-730 reversible arrow C-439 in alpha); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-beta, Y731- and Y730-alpha, and each protein was incubated with the appropriate second subunit beta(alpha), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y center dot) in the active alpha 2 beta 2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. H-2 electronnuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730, one to NH2Y731 and none to NH2Y356. Similar experiments with double mutants a-NH2Y730/C(439)A and alpha-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within a. The implications of these observations for the PCET process within a and at the interface are discussed.